Hemokinin-1 induces transcriptomic alterations in pain-related signaling processes in rat primary sensory neurons independent of NK1 tachykinin receptor activation

The tachykinin hemokinin-1 (HK-1) is involved in immunological processes, inflammation, and pain. Although the neurokinin 1 receptor (NK1R) is described as its main target, several effects are mediated by currently unidentified receptor(s). The role of HK-1 in pain is controversial, depending on the involvement of peripheral and central sensitization mechanisms in different models. We earlier showed the ability of HK-1 to activate the trigeminovascular system, but the mechanisms need to be clarified. Therefore, in this study, we investigated HK-1-induced transcriptomic alterations in cultured rat trigeminal ganglion (TRG) primary sensory neurons. HK-1 was applied for 6 or 24 h in 1 μM causing calcium-influx in these neurons, 500 nM not inducing calcium-entry was used for comparison. Next-generation sequencing was performed on the isolated RNA, and transcriptomic changes were analyzed to identify differentially expressed (DE) genes. Functional analysis was performed for gene annotation using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases. NK1R and Neurokinin receptor 2 (NK2R) were not detected. Neurokinin receptor 3 (NK3R) was around the detection limit, which suggests the involvement of other NKR isoforms or other receptors in HK-1-induced sensory neuronal activation. We found protease-activated receptor 1 (PAR1) and epidermal growth factor receptor (EGFR) as DE genes in calcium signaling. The transmembrane protein anthrax toxin receptor 2 (ANTXR2), a potential novel pain-related target, was upregulated. Acid-sensing ion channel 1; 3 (Asic1,3), N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors decreased, myelin production and maintenance related genes (Mbp, Pmp2, Myef2, Mpz) and GNDF changed by HK-1 treatment. Our data showed time and dose-dependent effects of HK-1 in TRG cell culture. Result showed calcium signaling as altered event, however, we did not detect any of NK receptors. Presumably, the activation of TRG neurons is independent of NK receptors. ANTXR2 is a potential new target, PAR-1 has also important role in pain, however their connection to HK-1 is unknown. These findings might highlight new targets or key mediators to solve how HK-1 acts on TRG.


. Introduction
Tachykinins represent a neuropeptide family widely distributed in the body.The first members of this family were substance P (SP), neurokinin A (NKA), derived from the preprotachykinin A/Tac1 gene, and neurokinin B (NKB), encoded by the preprotachykinin B/Tac3 gene (Borbély and Helyes, 2017).Other members of this family have also been identified: neuropeptide K (NPK) (Roth et al., 1985), neuropeptide γ (NPγ) (Kage et al., 1988), hemokinin-1 (HK-1), and endokinins (EKs) (Kurtz et al., 2002).HK-1 encoded by the preprotachykinin C/Tac4 gene was discovered 20 years ago in B lymphocytes (Zhang et al., 2000), and similar to SP, it was shown in several immune cell types, including myeloid (polymorphonuclear granulocytes and eosinophils), lymphoid, dendritic cells, neurons, and microglia (Borbély and Helyes, 2017).This widespread expression of HK-1 in diverse cell types might be related to a broad range of physiological and pathophysiological functions by activating a multitude of signaling pathways, which are currently not understood.HK-1 induces a variety of physiological and pathophysiological functions in the immune and hematopoietic systems, gastrointestinal tract, airways, cardiovascular, endocrine, and neural systems, bone, joints, and cancer.The role of HK-1 in pain is contradictory and likely to be concentration/dose dependent (Borbély and Helyes, 2017).In mice, centrally administered HK-1 caused scratching (Borbély and Helyes, 2017), licking, and biting at low doses (Watanabe et al., 2010).Intrathecally injected HK-1 was analgesic in nanomolar concentrations in an NK1-or opioid receptor-dependent manner, but hyperalgesic in picomolar concentrations (Fu et al., 2005).
Tachykinins possess a differential affinity for the three tachykinin receptors.HK and EKs have the highest affinity for NK1R, similar to SP (Satake et al., 2013;Steinhoff et al., 2014).They are all G protein-coupled receptors of the rhodopsin family.Signaling through the NK receptors is complex with multiple directions: (1) G q -related: activation of phospholipase C, resulting in inositol trisphosphate and diacylglycerol formation, mobilization of Ca 2+ from intracellular stores, and activation of protein kinase C; (2) G s -related activation of adenylyl cyclase, resulting in the cAMP formation and protein kinase A (PKA) activation; or (3) activation of phospholipase A2 and arachidonic acid production (Steinhoff et al., 2014;Garcia-Recio and Gascón, 2015).The existence of a specific receptor for HK-1 is currently intensively investigated (Duffy et al., 2003).It was shown that an NK1R antagonist did not inhibit pain caused by HK-1 in different concentrations, suggesting a specific HK-1 target/receptor (Watanabe et al., 2010).NK1R antagonists were developed as analgesic drug candidates, but they failed in human pain conditions.This might be due to different NK1R splice variants linked to distinct binding and activation mechanisms and/or a currently unidentified receptor (Zhang et al., 2000;Borbély and Helyes, 2017;Hunyady et al., 2019;Borbély et al., 2020).Our research group earlier showed that HK-1 mediated chronic adjuvant-induced inflammation and related pain (Borbély et al., 2013) and neuropathic and neurogenic inflammatory hyperalgesia via NK1 receptor activation (Hunyady et al., 2019).Interestingly, we recently demonstrated that unlike SP, HK-1 activates primary sensory neurons by inducing calcium influx via an NK1R-independent mechanism (Borbély et al., 2020).NK1R was shown to interact with the Mas-related G protein receptors (Mrgr) in certain pain-related mechanisms (Zhou et al., 2015).The human Mrgprx2, which is an ortholog of the rat Mrgprb5, was described as a potential target for HK-1 in mast cells to cause degranulation (Manorak et al., 2018).However, naturally occurring variants of Mrgprx2 lose HK-1 binding ability, showing the high importance of receptor variants (Alkanfari et al., 2018).
Based on a broad range of data demonstrating proinflammatory and pronociceptive functions of HK-1 in different organs partially independently of NK1 receptor activation (Hunyady et al., 2019;Borbély et al., 2020), our main aim was to identify the molecular mechanisms responsible for these actions.Our recent results demonstrated NK1R-independent calcium influx in cultured trigeminal ganglion cells induced by 1 µM, but not 500 nM HK-1 (Borbély et al., 2020).In the present study, we investigated the concentration-and treatment duration-dependent intracellular signaling mechanisms and pathways in these primary sensory neurons using an unbiased transcriptomic approach.
. Materials and methods

. . Primary cultures of TG neurons and treatment protocols
Primary cell cultures of TG neuron cells were extracted from 1-4-day-old Wistar rat pups as described earlier (Szoke et al., 2000).TG cells were dissected and washed in several steps.Afterward, TG cells were plated on poly-D-lysin-coated glass coverslips and grown in a nutrient-supplemented medium for the experiment.The earlier washing steps and the cell culture medium recipe are detailed elsewhere (Takács-Lovász et al., 2022).According to an earlier calcium influx, cell cultures were treated with HK-1 (Sigma Aldrich, solved in culture media) in two concentrations: 500 nM (no evoked calcium influx) and 1 µM [evoked calcium influx in an earlier study (Takács-Lovász et al., 2022)].Untreated cultures were used as controls.After 6 h and 24 h of HK-1 administration, samples were collected for RNA isolation.Except for the HK-1 500 nM 24 h condition, which was repeated in duplicates, other conditions were repeated in triplicate.

. . Illumina library preparation and sequencing
In order to provide a complete gene expression profile, RNA sequencing was performed (Fang and Cui, 2011).The library for Illumina sequencing was made using the QuantSeq 30 mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria).A total of 400 ng of total RNA was used for first-strand cDNA generation using an oligodT primer followed by RNA removal.The second strand synthesis was followed by random priming, and the products were purified with magnetic beads.Finally, the libraries were amplified and barcoded using PCR.All libraries were quality-checked on the TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA) to examine if adapter dimers formed during ./fnmol. .
PCR.The QuantSeq libraries were sequenced using the Illumina NextSeq550 platform to produce 75 bp single-end reads.

. . Bioinformatics
The sequencing reads were aligned against the Rattus norvegicus reference genome (Rnor 6.0 Ensembl release) with STAR v2.5.3a (Dobin et al., 2013).Following alignment, reads were associated with known protein-coding genes, and the number of reads aligned within each gene was counted using HTSeq library v0.11.1 (Anders et al., 2015).Gene count data were normalized using the trimmed mean of M values (TMM) normalization method of the edgeR R/Bioconductor package (v3.28,R v3.6.0,Bioconductor v3.9) (Robinson et al., 2010).Data were further logtransformed using the voom approach for statistical evaluation (Law et al., 2014) in the limma package (Ritchie et al., 2015).Fold change (FC) values between the compared groups resulting from the linear modeling process and moderated t-test p-values relative to a minimum required fold change threshold were calculated by the limma package.When determining differentially expressed (DE) genes, filtering thresholds were set to at least FC 1,2 and p-value 0.05 when the HK-1 1 µM 24 h and HK-1 500 nM 6 h treatments were compared to the untreated control group, and to FC 1.3 and p-value 0.05 for the HK-1 1 µM 6 h vs. untreated control, and to FC 2 and p-value 0.05 for HK-1 500 nM 24 h vs. untreated control group comparison.Normalized counts were represented as transcripts per million (TPM) values.Functional analysis (annotations of genes) was prepared using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome databases.Detection of functional enrichment was performed in the differentially expressed gene list (DE list enrichment: Fisher's exact test for GO, hypergeometric test for KEGG and Reactome) and toward the top of the list when all genes have been ranked according to the evidence for being differentially expressed (ranked list enrichment: non-parametric Kolmogorov-Smirnov test for GO and KEGG, hypergeometric test for Reactome) applying the topGO v2.37.0,ReactomePA v1.30.0, and gage v2.36.0 packages.The pathview package v1.26.0 (Luo and Brouwer, 2013) was used to visualize mapping data to KEGG pathways.GO terms were merged using the Revigo tool (tiny resulting list set up) for extracting the most relevant terms.

. . DE genes
Extracting the most important and relevant DE genes helps to understand the effects and intracellular signaling mechanisms of HK-1.In average rank, the p and FC values were taken into account shows the most relevant ranked gene list for all groups (Table 1).After 24 h of response to 1 µM HK-1, which we previously showed induces calcium influx in trigeminal primary sensory neurons, acid-sensing ion channel subunit 3 (Asic3), Glutamate ionotropic receptor NMDA type subunit 1 (Grin1), and C-C motif chemokine ligand 7 (Ccl7) were downregulated.At the earlier, 6 h timepoint, 1 µM HK-1 downregulated Slc25a5, while upregulating myelinassociated glycoprotein (Mag).For 6 h results, Mag, Itga4, and the LBH regulator of the Wnt signaling pathway (Lbb) were at the top of the list and upregulated independently of concentration.
The 500 nM HK-1 concentration, which did not evoke calcium influx in our earlier studies, downregulated Slc25a5 at 24 h and upregulated Ndufb6.Additionally, mitochondrially encoded NADH dehydrogenase 6 (Mt-nd6) was downregulated in the HK-1 500 nM 6 h group.Supplementary Images I1-I4 show heatmaps of all DE genes for different concentrations at different sampling times.
Similarly, altered DE genes over time (both at the 6 h and 24 h timepoints) in response to the same HK-1 concentration may yield insight into the key mechanisms and effects.Figure 1 shows the numerical representation of DE genes across all groups and a summary of different group comparisons.Most DE genes were detected in response to 500 nM HK-1 treatment after 24h, where Nr4a1, Slc25a5, F2r, Ndufb6, and Gnb2 were upregulated, which were confirmed by qPCR 1.783;2.205;2.105;1.445,respectively).Itga4, Fgf5, and Gnai1 were upregulated, and Ndufb6, Gnb2, and F2r were downregulated 6h after 1 µM HK-1 treatment, as determined both by sequencing and qPCR (FC: 2.942; 3.652; 1.14;−1.15;−1.01;−1.08).Unaltered genes were Fgfr1 and Gnai1 according to RNA sequencing and qPCR (FC: 1.385; 1.365) 24h after 1 µM HK-1 treatment.Although qPCR confirmed all these alterations obtained with sequencing, 24 h after 500 nM HK-1 treatment, Fgfr1 changes were different with the two   techniques: it was downregulated according to RNA sequencing but upregulated by PCR (FC: 1.18).The qPCR results are shown in Supplementary Table S2.
In response to 1 µM HK-1, the expression of three genes, Itga4, ANTXR cell adhesion molecule 2 (Antxr2), and teneurin transmembrane protein 3 (Tenm3), changed similarly (Figure 1).There was only one DE gene after 500 nM HK-1, Cxcl9 (C-X-C motif chemokine ligand 9), which was downregulated.Results for 24 h include less common DE genes, showing a greater concentration-dependent effect of HK-1.The common DE genes expressed in differently treated groups at the same timepoints showed 30 upregulated and 6 downregulated genes at 6 h and 3 upregulated and 5 downregulated genes at 24 h.

. . Potential targets for HK-
The mRNA level of the Tacr3 receptor was detected around the detection limit, showing that low expression of Tacr1 and Tacr2 was not found in these primary cell cultures.This result was confirmed by earlier, unpublished findings with primary cell cultures from adult rat trigeminal ganglia showing similar expression patterns of these receptors.The TPM values of these receptors in different primary cultures are shown in Supplementary Table S3.No significant difference was detected between the HK-1-treated and control groups; the overlapping receptors are shown in Figure 2. To identify potential target molecules, we collected relevant receptors related to neural and/or inflammatory mechanisms based on different gene databases.Notably, the MAS-related G proteincoupled receptor, member B5 (Mrgprb5), was detected in all groups, however, with transcripts per million (TPM) below 2. Figure 3 shows receptors present in control primary sensory neuronal cultures at 6 h and 24 h.Abbreviations for receptors can be found in Supplementary Table S4.The expressions of the receptors were similar at 6 h and 24 h.Rack1, Ngfr, and Ednrb were detected at very high TPM at both sampling timepoints.Tnfrsf12a, Adipor1, and Adipor2 were also present in both concentrations.Ntrk1, P2rx3, and F2r were also expressed in both cases, highlighting the possible impact on pain transmission and .
/fnmol. .calcium ion flow.Cxcr4 was found at 6 h. Figure 2 shows the DE receptors.Adipor2 at 500 nM 24 h was downregulated; F2r at 1 µM 6 h was, however, upregulated at 500 nM 24 h.Not only F2r but also Egfr expression was upregulated at 1 µM 6 h.An interesting result was the expression change in transient receptor potential melastatin 3,7,8 (Trpm3,7,8) cation channels at 500 nM 24 h.Supplementary Image I5 shows important receptors for all treatment conditions.

. . Signaling pathways influenced by HKtreatments
Significantly altered pathways 6 h after 500 nM and 1 µM HK-1 treatment determined by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database were predominantly related to calcium and Wnt signaling (Table 5).Figure 4 shows calcium signaling pathways with significant DE genes and represents the FC values of DE genes involved in this process.Among receptors, F2r had shown down-, Egfr upregulation.The transcriptomic level of Slc25a5 and Prkaca (protein kinase CAMP-activated catalytic subunit alpha) has changed negatively, and that of Gna11 (G protein subunit Alpha 11) and Prkacb (protein kinase cAMP-activated catalytic subunit beta) has changed positively.
The Gene Ontology (GO) database provides information on the functions of genes.Tables 6A-D shows the collected GO terms for all treated groups, simplified with the Revigo tool.Table 6A shows GO results for the HK-1 1 µM 6 h group.Protein kinase inhibitor activity, protein kinase A regulatory subunit binding, and thyroid hormone receptor binding are present in this list.Synaptic cleft was found to be a significant GO term in the 6 h results independent of the HK-1 concentration.Interestingly, biological processes altered by 500 nM HK-1 at 6 h (Table 6B) included the adenylate cyclase-activating G protein-coupled receptor signaling pathway, positive regulation of T cell-mediated immunity, neutrophil chemotaxis, positive regulation of leukocyte adhesion to vascular endothelial cells, and highlighting possible immunological functions.Notable cellular components were the synaptic cleft, T-tubule, and myelin sheath, and remarkable molecular functions were adrenergic receptor binding, NADH dehydrogenase activity, and cAMP binding.GO results revealed that 1 µM HK-1 treatment significantly altered the glutamate and insulin receptor signaling pathways at the 24-h timepoint, which are intracellular responses to calcium ions, as shown in Table 6C.
A notable affected cellular component was the synaptic vesicle.Among modified molecular functions, we found ligand-gated ion channel activity, postsynaptic neurotransmitter receptor activity, and sodium channel activity (Table 6C).Meanwhile, 500 nM HK-1 at the same timepoint regulated presynapse assembly, cGMPmediated signaling, mitochondrial ATP synthesis-coupled electron transport, Schwann cell proliferation, cell fate specification, positive regulation of dendritic spine development, positive regulation of cold-induced thermogenesis, and ceramide biosynthetic processes.As significant molecular functions, palmitoyltransferase activity, fibroblast growth factor binding, oxidoreductase activity, and acting on peroxide as an acceptor were found (Table 6D). .

. HK--induced signaling pathways
Table 7 shows results gained from the pathway database Reactome with DE genes for HK-1 1 µM 6 h, 500 nM 6 h, and 24 h groups.There was no significant finding for the HK-1 1 µM 24 h group.Noteworthy terms for HK-1 1 µM 6 h were apoptosis,    Significant column shows the number of DE genes from annotated genes of a given pathway.Pathway was altered significantly if a p-value of ≤ 0.05.Pathways, having a Significant score ≥ 4 were thought of as interesting findings.

FIGURE
Genes involved in the calcium signaling pathway from the KEGG database.Colored rectangle is for the altered expression of genes at h of HK-µM.Color means Z-score calculated from fold change value; green means downregulated, and red means upregulated.
Frontiers in Molecular Neuroscience frontiersin.orgsignal amplification, programmed cell death, G alpha (s) signaling events, chaperone-mediated autophagy, and opioid signaling.ADP signaling through P2Y purinoreceptor 12 and opioid signaling were also important results for HK-1 500 nM 6 h group.Additional relevant terms for HK-1 500 nM 6 h treatment group were oxidative stress-induced senescence and GABA receptor activation.
Common results for two concentrations at 6 h are the G alpha (s) signaling events, showing potential concentration-and calcium influx-independent effects of HK-1.Moreover, GPCR, GPCR ligand binding, and glycosphingolipid metabolism are important terms for HK-1 500 nM 6 h.

. Discussion
We demonstrate here the first transcriptomic data on the signaling pathways of HK-1 in rat primary sensory neurons, focusing on potential HK-1 targets, mechanisms of action, and DE genes involved in pain transmission and inflammation.The effect of HK-1 is complex, concentration dependent, and time dependent.Our findings support the well-described nociceptive actions of HK-1 and might explain the divergent neuronal activation processes.Since HK-1 is likely to act both on the primary sensory neurons and satellite glia cells, in this study we extract knowledge on these interactions to mimic in vivo conditions (Sakai et al., 2012;Theoharides et al., 2019).
Peripheral inflammatory and neuroinflammatory mechanisms resulting from complex interactions of immune cells, glial cells, and neurons play crucial roles in several diseases, including chronic pain (Siiskonen and Harvima, 2019).Itga4 and Antxr2 were upregulated for both concentrations, Tenm3 for 1 µM and Cxcl9 was downregulated after 500 nM at both timepoints.Itga4 encodes CD49d (alpha 4), one chain of very late antigen 4 (VLA-4), which belongs to adhesive molecules that activate the inflammatory process by facilitating the migration of immune cells into the central nervous system.The role of VLA-4 in genetic predisposition to chronic neuroinflammatory diseases has been demonstrated by several studies (Andreoli et al., 2007;Odoherty et al., 2007;Correia et al., 2009), supporting our findings in sensory neurons.Anthrax toxin components binding to ANTXR2, highly expressed on primary sensory neurons, were shown to inhibit inflammatory and neuropathic mechanical and thermal hyperalgesia (Yang et al., 2022).Tenm3 encodes a transmembrane protein also related to pain (Rouillard et al., 2016).Chemokines (CXCL4, CXCL9, CXCL10, and CXCL11) are important nociceptive mediators produced by neurons, microglia, and/or astroglia (Colvin et al., 2004).The CXCR3 receptor is expressed predominantly on T cells activated by CXCL9, CXCL10, and CXCL11 (Ransohoff, 2009).A CXCR3 receptor antagonist was recently shown to inhibit glia activation and neuropathic pain and enhance the effectiveness of morphine (Piotrowska et al., 2018).We showed the upregulation of Prss12, Mal, and Mag, but the downregulation of the Na v beta subunit 4 (Scn4b) 6 h after both HK-1 concentrations.Motopsyn/Prss12 can activate the PAR receptor in astrocytes and trigger glutamate release to activate neuronal NMDA receptors (Lee et al., 2007;Wójtowicz et al., 2015).MAL is a protein predominantly expressed by oligodendrocytes and Schwann cells and inhibits peripheral nerve myelination (Buser et al., 2009).Voltage-gated sodium channels (Na v ) initiate the action potential in excitable cells, and their mutations are implicated in chronic pain (Namadurai et al., 2015).
Figure 5 represents a summary of DE genes in different conditions.Both HK-1 concentrations resulted in more downregulated than upregulated genes at 24 h.Rph3a, Gabra2, Ryr2, Mag, and Scn1a were downregulated, while Hacd2 was upregulated.Hacd2 plays an important role in long-chain fatty acid biogenesis involved in neuronal membrane functions.Tachykinin receptors interact with Ryr2 in the endoplasmic reticulum, releasing calcium (Lin et al., 2005).The interaction of Rph3A with the NMDA receptor in hippocampal neurons plays a crucial role in synaptic retention and long-term potentiation.Moreover, Rph3A can also interact with AMPA receptors (Franchini et al., 2022).Notably, we have found remarkable upregulation of Fzd1 expressed by astrocytes and involved in their cross-talks (L'Episcopo et al., 2011(L'Episcopo et al., , 2018)).Increased levels of myelin-associated proteins, including Mag and Mbp proteins, were demonstrated through Activin A oligodendroglial ACVR1B-mediated white FIGURE Summary of calcium influx-related gene expression changes after µM HK-treatment.Red means upregulated, green means downregulated genes, and the gray arrow shows the -h e ect.Protein kinase A (PKA) was up and downregulated in di erent concentrations.Yellow rectangular means G α s signaling, and blue means G α q/ signaling.Gray rectangular demonstrates h result.Abbreviations: G protein subunit alpha I (GnaI), G protein subunit beta (GNB ), acid-sensing ion channel subunit , (Asic ; Asic ), potassium two pore domain channel subfamily K member (Kcnk ), taste receptor member (Tas r ), adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP), G protein subunit gamma (Gng ), and sodium voltage-gated channel beta subunit (SCN B) having a potential role in nociceptive sensation.
One of the most interesting issues is assigning potential targets for HK-1.Interestingly, the Tacr genes encoding tachykinin receptors were expressed around the detection limit in the primary sensory neuron cultures.These results are similar to the dorsal root ganglia data from Linnarson and co-workers (http://linnarssonlab.org/drg/).In human primary sensory neurons, Tac receptors were also detected at a low level (LaPaglia et al., 2018).Tachykinin receptors with three NK1R isoforms are expressed in primary sensory neurons of the rat trigeminal system (Beaujouan et al., 1999(Beaujouan et al., , 2002;;Page, 2005;Garcia-Recio and Gascón, 2015;Edvinsson et al., 2021), which we could not detect by our sequencing technique in the cultures derived from neonatal rats.The lack of a functionally active NK1 receptor in our system is supported by no calcium influx in response to SP treatment (Borbély et al., 2020).
HK-1 at 1 µM induced calcium signaling-related transcriptomic alterations 6 h later, which is supported by our earlier fluorescent calcium influx data, which was not inhibited either by the NK1 receptor antagonist CP99994 or NK1R deletion (Borbély et al., 2020).In calcium signaling processes, F2r and EGFR receptors were DEs (Figures 6, 7).F2r/PAR1 was downregulated, while the EGFR was upregulated.NK1R signaling by SP can activate tyrosine kinase receptors such as EGFR, and PAR1 can activate EGFR (Castagliuolo et al., 2000;Arora et al., 2008).This is supported by our earlier in vivo findings that in HK-1-deficient FIGURE FC value of significant DE genes at h of HK-µM involved in calcium signaling pathways.Red means upregulated genes and green shows downregulated genes.Abbreviations can be found in the Supplementary material.Asterisks denote these genes were found significant DE genes (*p < , ; **p < , ; ***p < , ) as analyzed by moderated t-test.
Our previous in vivo data demonstrated the pain-mediating role of HK-1 in several acute and chronic orofacial, neuropathic, and inflammatory pain models (partial sciatic nerve ligation, acute, and chronic CFA and mast cell tryptase-induced, as well as K/BxN serum-transfer arthritis models) (Borbély et al., 2013(Borbély et al., , 2020;;Aczél et al., 2018;Hunyady et al., 2019).Although it is difficult to directly compare the present results with in vivo conditions, several similarities can be determined between gene expressions in the mouse TRG after adjuvant-induced orofacial inflammation and the present TRG culture experiment following HK-1 treatment.We earlier showed that the HK-1 encoding Tac4 gene was upregulated in the TRG in the adjuvant-induced model.Meanwhile, fibroblast growth factor 13 (Fgf13, also termed as glia activating factor) was downregulated in wild-type mice, but upregulated in HK-1deficient ones (Aczél et al., 2020).This well correlates with the Fgf9 downregulation we observed in the present study in response to 1 µM 6 h later.Fgfs are involved in neuron-glia interactions and glia proliferation, mediating neuroinflammatory mechanisms (Stork et al., 2014).Furthermore, an orphan G protein-coupled receptor, Gpr62, playing a role in axo-myelinic signaling, was upregulated in the TRG of adjuvant-treated wild-type mice (Hay et al., 2021) similar to Gpr108 upregulation after 1 µM HK-1 in the present experiment to inhibit Toll-like receptor-triggered inflammatory responses (Dong et al., 2018).The voltage-gated K + channel-interacting protein 9 (Kcnj9) was also downregulated in the TRG after adjuvant injection (Aczél et al., 2020), which supports our findings on the Kcnip4 downregulations in the present model.Inward-rectifying K+ currents have been shown to change in the trigeminal ganglia during peripheral inflammation (Takeda et al., 2011).Transmembrane protein 100 (Tmem100) was upregulated in the TRG in the in vivo mouse model, while in our cell system the expressions of related members of this protein family, Tmem128, significantly increased.Integrin subunit alpha 7 (Itga7) involved in glia proliferation (Tan et al., 2022), was also downregulated both in the mouse model (Aczél et al., 2020) and in the present cell culture study Itgav both are expressed on glial cells (Mapps et al., 2022).RNA-Seq literature data demonstrated Kcnj9, F2rl2 (PAR3) upregulation in the DRG in a neuropathic pain model similar to what we found for F2r and PAR1 (Stevens et al., 2020).Validation of the RNA sequencing data with qPCR showed a good correlation for all investigated genes except Fgfr1.However, such discrepancies are not unusual based on the literature data; others described that these two techniques provide similar results in 87.9% (Protasio et al., 2013) and 95.2% of genes (Liu et al., 2014), which is in agreement with the present findings.
In conclusion, this is the first approach to determining transcriptomic alterations induced by HK-1 in primary sensory neurons that might be related to their activation mechanisms and pain.We showed concentration-and time-dependent actions and identified potential pain-related processes such as microglialneuron or GPCR interactions, myelin-associated gene expression, and orphan GPCRs having roles in GABAergic pathways by modifying NMDA and AMPA receptor subunits, demonstrating that not just neurons, but mast cells and glia cells might be affected by HK-1.The effects specific to the 1 µM (sodium channel activity, neurotransmitter receptor activity, cytokine activity) were potentially calcium influx-dependent, as we earlier showed that the 1 µM, but not the 500 nM concentration, induced a calcium signal in this experimental paradigm.Although the target(s) of HK-1 cannot be determined on the basis of the present results, it is not likely to be the NK1 tachykinin receptor.Identifying the receptor might open novel perspectives in analgesic research.

FIGURE
FIGURE Number of DE genes for all groups.Insert tables show the FC values of the DE genes for the respective comparisons.(A) Panel demonstrates the upregulated genes and (B) shows the numbers of downregulated genes.

FIGURE
FIGURETPM values of receptors in control primary sensory neuronal cultures h and h.All data are shown in the Supplementary Image I .Receptors having a TPM > with specific roles in neural mechanisms are demonstrated here.Functions of receptors were filtered based on public databases (https://rgd.mcw.edu/,https://www.genecards.org/, and https://www.ncbi.nlm.nih.gov).

FIGURE
FIGUREFC value of significant di erentially expressed genes in primary sensory neuron cultures of the trigeminal ganglia in response to two HKconcentrations after h and h treatment durations.Abbreviations are shown in Supplementary TableS.All DE genes can be found in Supplementary Images I -I .

FIGURE
FIGURESummary of DE genes found in di erent treatment conditions: red columns refer to the respective group comparisons and the black columns demonstrate the shared DE genes of two or more groups.H: HK-.

TABLE Collection
TABLE Common DE genes for the µM and nM HK-concentrations at h. Red means upregulated, while green shows downregulated DE genes.
TABLE Common DE genes for both doses at h. Red means upregulated DEs, whereas green shows downregulated Des.

TABLE FC ,
p, and avgRank values for other possible relevant and significant DE genes for all groups.

TABLE A -
D. GO terms for each group merged with the Revigo tool.

TABLE Reactome
Results were relevant and significant, if FDR < 0.25 and a p-value of < 0.05.The total number of background genes was 3890.Terms are in order according to p increasing value showing less statistical significance.Date ratio shows DE genes/genes involved in the Reactome pathway.